Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes

Dziegala,Magdalena; Kasztura,Monika; Kobak,Kamil; Bania,Jacek; Banasiak,Waldemar; Ponikowski,Piotr; Jankowska,Ewa A.

doi:10.3892/mmr.2016.5705

Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes

Authors:
- Magdalena Dziegala
- Monika Kasztura
- Kamil Kobak
- Jacek Bania
- Waldemar Banasiak
- Piotr Ponikowski
- Ewa A. Jankowska
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Affiliations: Students' Scientific Organization, Department of Heart Diseases, Wrocław Medical University, 50‑367 Wrocław, Poland, Department of Heart Diseases, Wrocław Medical University, 50‑367 Wrocław, Poland, Laboratory for Applied Research on Cardiovascular System, Department of Heart Diseases, Wrocław Medical University, 50‑981 Wrocław, Poland, Department of Food Hygiene and Consumer Health, Wrocław University of Environmental and Life Sciences, 50‑375 Wrocław, Poland, Centre for Heart Diseases, Military Hospital, 50‑981 Wrocław, Poland
Published online on: September 5, 2016 https://doi.org/10.3892/mmr.2016.5705
Pages: 3969-3977

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Abstract

The differential availability of iron during hypoxia is presumed to affect the functioning of cardiac and skeletal myocytes. Rat H9C2 cardiomyocytes and L6G8C5 myocytes were cultured for 48 h in normoxic or hypoxic conditions at the optimal, reduced or increased iron concentration. The mRNA expression levels of markers of apoptosis [B‑cell lymphoma‑2 (Bcl2; inhibition) and Bcl‑2‑activated X protein (Bax; induction)], atrophy (Atrogin), glycolysis (pyruvate kinase 2; PKM2) and iron metabolism [transferrin receptor 1 (TfR1; iron importer), ferroportin 1 (FPN1; iron exporter), ferritin heavy chain (FTH; iron storage protein) and hepcidin (HAMP; iron regulator)] were determined using reverse transcription‑quantitative polymerase chain reaction, and cell viability was measured using an tetrazolium reduction assay. Cardiomyocytes and myocytes, when exposed to hypoxia, demonstrated an increased Bax/Bcl‑2 gene expression ratio (P<0.05). Additional deferoxamine (DFO) treatment resulted in further increases in Bax/Bcl‑2 in each cell type (P<0.001 each) and this was associated with the 15% loss in viability. The analogous alterations were observed in both cell types upon ammonium ferric citrate (AFC) treatment during hypoxia; however, the increased Bax/Bcl‑2 ratio and associated viability loss was lower compared with that in case of DFO treatment (P<0.05 each). Under hypoxic conditions, myocytes demonstrated an increased expression of PKM2 (P<0.01). Additional DFO treatment caused an increase in the mRNA expression levels of PKM2 and Atrogin‑1 (P<0.001 and P<0.05, respectively), whereas AFC treatment caused an increased mRNA expression of PKM2 (P<0.01) and accompanied decreased mRNA expression of Atrogin‑1 (P<0.05). The expression augmentation of PKM2 during hypoxia was greater upon low iron compared with that of ferric salt treatment (P<0.01). Both cell types upon DFO during hypoxia demonstrated the increased expression of TfR1 and HAMP (all P<0.05), which was associated with the increased Bax/Bcl‑2 ratio (all R>0.6 and P<0.05). In conclusion, during hypoxia iron deficiency impairs the viability of cardiomyocytes and myocytes more severely compared with iron excess. In myocytes, during hypoxia iron may act in a protective manner, since the level of atrophy is decreased in the iron‑salt‑treated cells.

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